During the development of obesity, an altered diet leads to changes in the gut microbiota, including decreased diversity and relative abundance of a protective microbe, Akkermansia muciniphila. This leads to a pro- inflammatory gut environment, inflammation in distant tissues (i.e., adipose), accumulation of fat and eventually insulin resistance (IR). Current rodent models of fat accumulation focus on one etiological cause and use in- bred laboratory strains; however, translation of these studies to human medicine has been limited, presumably because a) humans are a more heterogeneous outbred population with a more variable microbiota than laboratory rodents, and b) the development of obesity and IR results from a combination of dietary, metabolic and genetic factors. Hibernating mammals like 13-lined ground squirrels (Ictidomys tridecemlineatus) naturally develop adiposity each year during a ?pre-hibernation fattening phase?. During this phase they undergo microbiota and immune changes similar to that observed in obese humans and develop IR. Additionally, ground squirrels are, like humans, an outbred, genetically diverse population in which adiposity results from a variety of host and environmental factors. In combination this suggests that hibernators are an excellent model for examining the role of the microbiota and immune system in fat accumulation, with results that are highly translatable to human health. Therefore, the goal of this study is to elucidate the role of these parameters in pre-hibernation fattening in ground squirrels and to discover whether manipulations of these parameters affect the rate and degree of fattening and the development of IR. The hypotheses for this investigation are that 1) changes in the composition and function of the gut microbiota promote fat accumulation and development of IR during pre-hibernation fattening and 2) the intestinal immune system shifts to a pro- inflammatory state during the pre-hibernation fattening period which is associated with metabolic inflammation, fat accumulation and IR. The proposed experimental approach uses manipulations of the gut microbiota and intestinal immune system of ground squirrels during their pre-hibernation fattening period to determine the role of the microbiota and intestinal immune system on the development of obesity and IR. Through manipulation of the microbiota via introduction of lean-season microbiota, Specific Aim 1 will examine the role of microbiota structure and function on the development of obesity and IR in ground squirrels. In Specific Aim 2 we will treat with a gut-specific anti-inflammatory drugs to evaluate the role of inflammatory changes on pre-hibernation fattening in ground squirrels. Our experiments promise to illuminate the role of the gut microbiota and immune system in the accumulation of fat and development of IR that occurs annually in this species, ultimately contributing to our overall knowledge of how fat accumulation and IR are induced in these animals. Proper development of this model will lead to further use of ground squirrels as an outbred, genetically diverse model in which to study treatments that can help treat human obesity.